This invention relates to nucleic acid and amino acid sequences of a human ankyrin family protein and to the use of these sequences in the diagnosis, treatment, and prevention of autoimmune/inflammatory, cell proliferative, and vesicle trafficking disorders.
Cells contain a cytoskeleton that links intracellular compartments with each other and the plasma membrane. Associations between the cytoskeleton and the lipid membranes bounding these compartments involve spectrin, ankyrin, and integral membrane proteins. Spectrin is a major component of the cytoskeleton and acts as a scaffolding protein. Similarly, ankyrin acts to tether the actin-spectrin moiety to membranes and to regulate the interaction between the cytoskeleton and membranous compartments. Different ankyrin isoforms are specific to different organelles and provide specificity for this interaction. Ankyrin also contains a regulatory domain that can respond to cellular signals, allowing remodeling of the cytoskeleton during the cell cycle and differentiation (Lambert, S. and Bennett, V. (1993) Eur. J. Biochem. 211:1-6).
Ankyrins have three basic structural components. The N-terminal portion of ankyrin consists of a repeated 33-amino acid motif, the ankyrin repeat, which is involved in specific protein-protein interactions. Variable regions within the motif are responsible for specific protein binding, such that different ankyrin repeats are involved in binding to tubulin, anion exchange protein, voltage-gated sodium channel, Na+/K+-ATPase, and neurofascin. The ankyrin motif is also found in transcription factors, such as NF-xcexa-B, and in the yeast cell cycle proteins CDC10, SW14, and SW16. Proteins involved in tissue differentiation, such as Drosophila Notch and C. elegans LIN-12 and GLP-1, also contain ankyrin-like repeats. Lux et al. (1990; Nature 344:36-42) suggest that ankyrin-like repeats function as xe2x80x98built-inxe2x80x99 ankyrins and form binding sites for integral membrane proteins, tubulin, and other proteins.
The central domain of ankyrin is required for binding spectrin. This domain consists of an acidic region, primarily responsible for binding spectrin, and a basic region. Phosphorylation within the central domain may regulate spectrin binding. The C-terminal domain regulates ankyrin function. The C-terminally-deleted ankyrin, protein 2.2, behaves as a constitutively active ankyrin, displaying increased membrane and spectrin binding. The C-terminal domain is divergent among ankyrin family members, and tissue-specific alternative splicing generates modified C-termini with acidic or basic characteristics (Lambert, supra).
Three ankyrin proteins, ANK1, ANK2, and ANK3, have been described which differ in their tissue-specific and subcellular localization patterns. ANK1, erythrocyte protein 2.1, is involved in protecting red cells from circulatory shear stresses and helping maintain the erythrocyte""s unique biconcave shape. An ANK1 deficiency has been linked to hereditary hemolytic anemias, such as hereditary spherocytosis (HS), and a neurodegenerative disorder involving loss of Perkinje cells (Lambert, supra). ANK2 is the major nervous tissue ankyrin. Two alternative splice variants are generated from the ANK2 gene. Brain ankyrin 1 (brank1), which is expressed in adults, is similar to ANK1 in the N-terminal and central domains, but has an entirely dissimilar regulatory domain. An early neuronal form, brank2, includes an additional motif between the spectrin-binding and regulatory domain. An ankyrin homolog in C. elegans, unc-44, produces alternative splice variants similar to ANK2. Mutations in the unc-44 gene affect the direction of axonal outgrowth (Otsuka, A. J. et al. (1995) J. Cell Biol. 129:1081-1092).
ANK3 consists of four ankyrin isoforms (G100, G119, G120, and G195), which localize to intracellular compartments and are implicated in vesicular transport. AnkG119 is associated with the Golgi, has a truncated N-terminal domain, and lacks a C-terminal regulatory domain. AnkG120 and AnkG100 associate with the late endolysosomes in macrophage, lack N-terminal ankyrin repeats, but contain both spectrin-binding and regulatory domains characteristic of ANK1 and ANK2. AnkG195 is associated with the trans-Golgi network (TGN). These ankyrin isoforms are part of a spectrin complex which may mediate transport of proteins through the Golgi complex. A spectrin-ankyrin-adapter protein trafficking system (SAATS) has been proposed for the selective sequestration of membrane proteins into vesicles destined for transport from the ER to the Golgi and beyond. In this model, intra-Golgi, TGN, and plasma membrane transport would involve exchange of SAATS protein components, including ankyrin isoforms, to specify and distinguish the final destination for vesicular cargo (DeMatteis, M. A. and Morrow, J. S. (1998) Curr. Opin. Cell Biol. 10:542-549).
The discovery of a new human ankyrin family protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of autoimmune/inflammatory, cell proliferative, and vesicle trafficking disorders.
The invention is based on the discovery of a new human ankyrin family protein (ANFP), the polynucleotides encoding ANFP, and the use of these compositions for the diagnosis, treatment, or prevention of autoimmune/inflammatory, cell proliferative, and vesicle trafficking disorders.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of ANFP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of ANFP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.